The mechanisms involved in gene expression and regulation have been intensively studied in a variety of cells and organisms. These analyses have indicated that numerous elements can play an important role in the constitutive or differential expression of genes within an organism, cell, or during development from embryo to adult. Various factors and processes acting in a coordinate fashion modulate transcription in either a positive (activation) or negative (repression) manner. Some of these elements act in cis and are localized to the intergenic spacer regions; others act in trans and are themselves the products of regulated templates. While these elements may directly affect transcription, other cellular events which occur co- or posttranscriptionally may also exert either a positive or negative affect on expression, regulation, and ultimately development. Little is known about the co- or posttranscriptional events which are instrumental in the expression and regulation of plasmodial genes during schizogony or sporogony. Therefore, the overall goal of the current proposal is to elucidate possible regulatory pathways involved in differential expression of the P. falciparum genome during early and late stage asexual erythrocytic development. The regulation of two differentially expressed templates, representing early" (the knob-associated histidine-rich protein a KAHRP) and "late" (the major merozoite surface antigen MMSA a pf2OO/pl95) genes, will be investigated. The results obtained from these analyses may provide some insight into the overall mechanisms of cellular differentiation in plasmodia. Determine the temporal order of transcription of the KAHRP (early) and pf2OO (late) genes. Studies to determine this will include northern blot analysis of synchronous cultures and immunoprecipitation of radiolabelled proteins. (NOTE: Part of these studies have been included in Section C. Preliminary Studies). Determine if co- and/or posttranscriptional events (splicing, mRNA stability, multiple transcription initiation sites, nuclear export, polyadenylation) play a role in differential expression of these genes. These analyses will employ hybrid selection of temporally expressed mRNA; actinomycin D treatment of synchronous populations of parasitized erythrocytes to determine mRNA stability/decay rates; capping analyses to determine if caps are present and their degree of methylation; splicing of the KAHRP intron to determine if intron retention is implicated in regulation; northern blot analysis of nuclear and cytoplasmic fractions to determine if regulation occurs at the level of export from the nucleus. Primer extension studies will also be undertaken to determine if 5' terminal heterogeneity, an indication that multiple promoters or transcription initiation start sites, are utilized by these transcripts, and are employed as a means to regulate mRNA gene expression.